We show that the dynamics of proteins-NP interactions are differently mediated by different cellular media, which are the liquid environments where NPs encounter cells. We used, as a model, spherical NPs (citrate-capped gold nanoparticles, AuNPs) of different sizes (15, 40 and 80 nm). Upon incubation with two widely used cellular media (i.e., DMEM and RPMI, supplemented with fetal bovine serum), we analyzed NPs interactions with serum proteins present in the media, over time, by several spectroscopic techniques (DLS, UV-Vis, PRLS). We found that, while DMEM elicited the formation of a large time-dependent protein corona, RPMI shows different dynamics with a reduced protein coating. Nature and composition of the hybrid bio-nanostructures were then characterized by ex-situ investigations. Finally, regarding their biological impact, we found out that the different protein coating onto the NPs surfaces, producing hybrid complexes of diverse size, exert different toxicity. In particular, by applying label-free 2-photon confocal microscopy and ICP-MS, we demonstrated that smaller complexes, in RPMI solution, are more abundantly up-taken by cells, exerting higher toxicity.